This invention relates to gel electrophoresis and more particularly to a method and apparatus for gel electrophoresis which employs periodic inversion of the electric field.
Electrophoresis involves the separation of mixtures by differential migration of components through a transport medium in an electric field. Many particles in aqueous solution acquire an electrical charge due to ionization and thus move in response to an external electric field. The charged particles may be simple ions, complex macromolecules, viruses, colloids or even living cells. The rate of their migration depends upon the amount of charge, the size and shape of the particle, and the properties of the solvent.
Electrophoresis in a gel medium is an important method of separating proteins, nucleic acids and other such macromolecules in mixture. When an electric field is applied to the medium at a given pH, the macromolecules migrate toward the oppositely charged electrode. The higher their ratio of charge to mass, the faster they move. The mixture of macromolecules is thereby eventually separated into a series of distinct bands in order of charge density. The electrophoresis is generally terminated when the leading band has migrated through most of the available gel. The bands can be identified by suitable means such as staining, optical scanning and the like procedures, and the macromolecules can be recovered by cutting out and solubilizing the corresponding portions of the gel. This can be done, for example, by electroelution from the gel or by chemical or physical disruption of the gel structure followed by appropriate purification techniques.
Agarose, which is a naturally occurring linear polysaccharide of galactose and 3,6-anhydrogalactose, is particularly useful as the electrophoretic support medium since it permits the separation of very large molecules such as viruses, enzyme complexes, lipoproteins and nucleic acids which are often outside the useful pore size with polyacylamide gel electrophoresis. A large variety of agaroses and modified agaroses are available commercially. They are usually used in concentrations ranging from about 0.1 to about 2.5% by weight.
Notwithstanding the foregoing, the use of conventional agarose gel electrophoresis has not generally been ideally suited for separation of the largest deoxyribonucleic acid (DNA) molecules, that is, molecules which are larger than about 2.times.10.sup.5 base pairs (bp). Most practical work has been confined to molecules less than about 2.times.10.sup.4 bp. Although typical DNA molecules employed in genetic engineering applications are within this lower size range, the DNA molecules in chromosomes are larger.
Further background information on conventional gel electrophoresis of DNA can be had by reference to a text such as Rickwood and Hames, Gel Electrophoresis of Nucleic Acids: A Practical Approach, IRL Press, Oxford, UK, particularly Chapter 2, "Gel Electrophoresis of DNA", by Sealey and Southern.
For background information on attempts to achieve separation of very large DNA molecules by conventional gel electrophoresis, reference can be had to papers by Fangman, Nucleic Acids Res. 5, 653-665 (1978); and Serwer, Biochemistry 19, 3001-3004 (1980). In the former paper, using very dilute agarose gels (which are difficult to handle) and low voltages (which require long running times), Fangman was able to achieve a mobility ratio of bacteriophage G DNA (approximately 750 kb, where 1 kb=1 kilobase pair=1000 base pairs) to bacteriophage T4 DNA (approximately 170 bp) of approximately 1.4. Molecules larger than bacteriophage G were not investigated. So also in the latter paper, Serwer found that the best conditions involved dilute agarose gels run at low voltages. Molecules larger than approximately 170 kb were not investigated.
Recently, a modified gel electrophoresis technique for separating large DNA molecule was disclosed by Schwartz et al., Cold Spring Harbor Symp. Quant. Biol. 47, 189-195 (1983); Schwartz and Cantor, Cell 37, 67-75 (1984); and Cantor and Schwartz, U.S. Pat. No. 4,473,452. According to their disclosed technique, the DNA molecules are separated by subjecting the gel medium to two non-uniform electric fields having co-planar directions which are transverse to each other. The DNA molecules thereby migrate in a direction that lies in between the two field directions. Although the disclosed Cantor and Schwartz technique has been applied with success to separate DNA molecules present in the chromosomes of lower organisms such as yeast and protozoans, the bands are somewhat distorted and nonparallel, whereby it is difficult to make lane-to-lane comparisons between samples as is obtained in conventional gel electrophoresis. Moreover, the transverse-field gel electrophoresis technique requires complex electrode geometries. Although the theoretical minimum is three, no devices have been described that contain fewer than four, and it is common for devices to feature whole arrays of electrodes. Furthermore, the precise positioning of the electrodes has dramatic effects on the results obtained. Consequently, transverse-field-alternation gel electrophoresis does not provide for convenient gel electrophoretic practice.
Implementation of the transverse-field technique (also defined as orthogonal-field-alternation) and applications to the chromosomal DNA molecules from yeast are described by Carle and Olson, Nucleic Acids Res. 12, 5647-5664 (1984). A description of the complete analysis of the set of chromosomal DNA molecules from yeast using the transverse-field technique is further reported by Carle and Olson, Proc. Natl. Acad. Sci. U.S.A. 82, 3756-3760 (1985).
Other background information on the application of the transverse-field technique of gel electrophoresis to chromosomal DNA molecules is provided by Van der Ploeg et al., Cell 37, 77-84 (1984); Van der Ploeg et al., Cell 39, 213-221 (1984); and Van der Ploeg et al., Science 229, 658-661 (1985).